JCS033803 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Retrograde movement of the organelles of bulk endocytosis. Growth cone (2 DIV) imaged either immediately after loading with FM4-64 (1 minute in KRH, red) or after a 6-minute incubation at 37°C in KRH. Endocytic organelles containing FM4-64, initially concentrated in the T domain of the growth cone, move retrogradely along the axon. Bar, 10 µm.

• Supplemental Figure S2 -

  Fig. S2. Bead uptake by bulk endocytosis is sensitive to F-actin disruption, PI3-kinase inhibition and cholesterol depletion. Growth cones were loaded with 20-nm beads (red) during incubation in KRH for 1 minute. (A) Top panels: imaging of a growth cone before and 5 minutes after bead loading. Bead internalization is followed by extension of preexisting short filopodia (asterisks). Middle and bottom panels: CytD (10 µM, 15 minutes), LY294002 (50 µm, 30 minutes) and MβCD (5 mM, 3 minutes) inhibit bead uptake and filopodia extension. Both processes were restored after incubation of MβCD-treated neurons for 30 minutes in KRH at 37°C (recovery). (B) Acute (3 minutes) MβCD application efficiently reduces cholesterol levels in growth cones compared with control growth cones (resting), as assessed by filipin staining (blue). Normal cholesterol levels are restored after incubation in KRH for 30 minutes (recovery).

• Supplemental Figure S3 -

  Fig. S3. Representative images of growth cones of DIV3 neurons coexpressing YFP (green in the merge images) and either the dominant-negative mutant Rac1-N17 (top panel) or the constitutively active mutant Rac1-V12 (middle panel), or expressing only YFP (bottom panel), incubated in KRH containing FM4-64 (red in the merge images) for 1 minute.

• Movie 1 -

  Movie 1. Phase-contrast videomicroscopy of the growth cones shown in Fig. 2, followed by the corresponding fluorescent image after FM4-64 loading (1 minute, KRH) and fixation. Endocytosis-negative growth cone.

• Movie 2 -

  Movie 2. Phase-contrast videomicroscopy of the growth cones shown in Fig. 2, followed by the corresponding fluorescent image after FM4-64 loading (1 minute, KRH) and fixation. Endocytosis-positive growth cone.